The long term goal of this work is to understand the basic mechanisms involved in the control and catalysis of pre-mRNA splicing by the spliceosome. In particular, we are interested in understanding the mechanism of splicing catalysis and interactions between the components of the catalytic center. We focus our efforts on elucidating the molecular mechanisms involved in positioning of the 5' splice site (5'SS) for catalysis. In this proposal we describe experiments focusing on the role of two spliceosomal proteins in this process: Prp8p and Prp28p. Previous biochemical and genetic studies strongly suggested that Prp8p makes important functional contacts with both splice sites and U6 snRNA within the catalytic core of the complex. We plan to analyze these interactions at the molecular level, through a series of RNA binding and crosslinking studies using recombinant fragments of Prp8p. We will also test these recombinant Prp8p fragments for the ability to support splicing of appropriate RNA substrates. Finally, using both biochemical and genetic strategies, we will analyze interactions between Prp8p and Prp28p, since we expect that these proteins are positioned in close proximity of each other in the spliceosome. Using a combination of genetic and biochemical approaches, we will analyze the role of hPrp28p in selection of the 5'SS. We will also continue to characterize spliceosomal proteins (in particular p27) located in proximity of the 5'SS and hPrp28p. Finally, we will analyze contacts made by the 5'SS within the spliceosome prior to the ATP-dependent step in complex assembly. Together, these studies will provide a better understanding of the structure of the spliceosome and the role of Prp28p in its formation and function.